Effect of NFAT isoform-specific knockdown on TNFα-induced PPFC cell adhesion adhesion

In order to assess the functional effects of isoform-specific changes in TNFα-induced HRMEC, monolayers were transfected with either control or isoform-specific

0

Control Control NFATc1 NFATc2 NFATc3 NFATc4

Secreted Protein (pg/mg)

Control Control NFATc1 NFATc2 NFATc3 NFATc4

Secreted Protein (pg/mg)

Control Control NFATc1 NFATc2 NFATc3 NFATc4

Secreted Protein (pg/mg)

Control Control NFATc1 NFATc2 NFATc3 NFATc4

Secreted Protein (pg/mg)

36

siRNA and evaluated using a PPFC cell adhesion assay. TNFα treatment (1ng/ml for 4hrs) of monolayers transfected with control siRNA induced an 11.5-fold increase in PBMC adhesion compared to non-stimulated controls (p<0.0001, Figure 14).

Monolayers transfected with NFATc2 and NFATc4 siRNA reduced TNFα-induced PBMC adhesion by 55% (p=0.0013) and 38.4% (p=0.0289), respectively, while transfection with NFATc1 and NFATc3 siRNA had no significant effect on TNFα-induced cell adhesion.

Figure 14. Effect of isoform-specific siRNA knockdown on TNFα-induced PBMC adhesion. HRMEC monolayers transfected with either control or isoform-specific siRNA were treated with TNFα (1ng/ml) for 4hrs. Monolayers transfected with NFATc2 and NFATc4 siRNA significantly reduced TNFα-induced PBMC adhesion. Each bar represents the mean ± SEM (n=5). * = p<0.05, ** = p<0.01

0 20 40 60 80 100 120 140 160

Control Control NFATc1 NFATc2 NFATc3 NFATc4

Average Cells / mm2

TNFα-Induced PBMC Adhesion

**

*

siRNA:

TNFα (1ng/ml)

37 3.3 Conclusions

The previous study revealed a general role for NFAT signaling in the response of HRMEC to TNFα-stimulation, and identified a functional role in TNFα-induced cell adhesion (Chapter II). The present study builds upon those early findings, using isoform-specific siRNA to identify new, discreet, and at times counteractive roles for the individual NFAT isoforms in this context.

The identification and verification of isoform-specific siRNA was a critical first step in being able to evaluate the role of individual NFAT isoforms. At the start of this study, no protocol had been established for the effective transfection of primary human retinal microvascular endothelial cells, and early efforts using Lipofectamine® (Life Technologies; Carlsbad, CA), DharmaFECT (GE Healthcare; Little Chalfont, U.K), and Polyjet™ (SignaGen® Laboratories, Rockville, MD) techniques proved unsuccessful.

Eventually, a successful protocol was adapted using a combination of Virofect and Targafect reagents (6.2.1), and oligos targeting the individual isoforms could be evaluated.

Considerable effort was put into identifying oligos that could effectively knockdown the target isoform at a low concentration (75nm) and with minimal effect on other isoforms. Table 8 identifies the oligos identified for each target and used in subsequent studies. In the end, greater than 70% knockdown was achieved for all of the isoforms, and only NFATc3 expression was affected by siRNA targeting a different isoform. In this instance both NFATc2 and NFATc4 siRNA knocked down expression of NFATc3 by roughly 30%. This was not due to an overlap in the target sequence.

NFATc2 has previously been shown to play a role in the expression of other NFAT

38

isoforms, and this is believed to be responsible for the effect seen here, though there are no reports to date that have evaluated the role of either NFATc2 or NFATc4 in NFATc3 expression. In both cases, it is believed that this minimal reduction had no

effect on the subsequent studies, as NFATc2 and NFATc4 knockdown both caused significant effects that were counter to those seen by the more significant NFATc3 knockdown via NFATc3 siRNA.

Having established the appropriate techniques and reagents to evaluate the roles of individual NFAT isoforms, isoform-specific siRNA was used to further elucidate the contributions of NFAT signaling in TNFα-induced gene expression. The first two targets analyzed were CX3CL1 and VCAM1, which had been identified as NFAT-regulatory targets in the RNA-seq study. Isoform-specific siRNA revealed that NFATc2 knockdown negatively regulated both CX3CL1 and VCAM1 expression, and that NFATc3 knockdown increased TNFα-induced upregulation of CX3CL1. The latter finding showed that individual NFAT isoforms can play counteractive roles in endothelial cell activities, and highlights the importance of evaluating isoforms individually.

Given these initial findings, the study was expanded to examine the role of NFAT isoforms in TNFα-induced SELE and ICAM1 expression. SELE is the gene that codes for E-selectin, and both E-selectin and ICAM1 are adhesion proteins known to mediate TNFα-induced leukocyte adhesion. While the previous study did not find any significant effect of NFAT inhibition on TNFα-induced expression of either target, investigators working in other endothelial cell systems have identified a regulatory role for NFAT in the expression of both.103,148,155,167 Isoform-specific siRNA knockdown showed that this incongruity was due to counteracting effects of individual isoforms that were masked by

39

inhibition of all four calcineurin-dependent isoforms with INCA-6. Both NFATc2 and NFATc4 knockdown significantly reduced TNFα-induced expression of SELE, while NFATc3 increased its expression. In the case of ICAM1 expression, NFATc2 again inhibited TNFα-induction and NFATc1 knockdown exacerbated the induction.

The previous analyses using INCA-6 also highlighted a role for NFAT signaling in the regulation of TNFα-induced chemokines CXCL10 and CXCL11. CXCL10 and CXCL11 both serve as ligands for the CXCR3 receptor and are known to play a role in leukocyte recruitment to sites of endothelial inflammation.^79-81 While this activity would not be expected to manifest functionally in an in vitro PPFC model for TNFα-induced leukocyte adhesion, leukocyte recruitment is a critical feature of TNFα-induced leukostasis in vivo. Accordingly, the effect of isoform-specific siRNA on TNFα-induced protein secretion for each of these cytokines was measured. In this context, NFATc4 siRNA proved to be the most potent regulator of TNFα-induced cytokine secretion, inhibiting both CXCL10 and CXCL11 levels in conditioned media. As was the case with E-selectin and ICAM1, MCP-1 and IL-6 are inflammatory products known to be upregulated by TNFα-stimulation and identified as NFAT-regulatory targets in other endothelial cell populations.^150,167 As such, they were included in this study, and NFATc4 siRNA was again found to reduce TNFα-induced levels in the case of MCP-1, though TNFα-induced secretion of IL-6 was unaffected by all isoforms. Taken together, these findings (summarized in Table 6) identify a strong role for NFATc2 in the expression of TNFα-induced leukocyte adhesion proteins, as well as NFATc4 in the production of TNFα-induced leukocyte chemoattractants. Interestingly, a separate study evaluating the role of individual NFAT isoforms in VEGF-induced cytokine production, found that

40

NFATc1 siRNA reduced VEGF-induced IL-6 levels (Appendix B), indicating that not only is the relationship between NFAT and a particular target stimuli dependent, but that the isoforms involved may be as well.

TNFα-Induced

In order to again test whether the observed findings had a functional impact on TNFα-induced leukocyte adhesion, the effect of isoform-specific siRNA was evaluated in a PPFC assay. The previous study using this technique showed that pharmacologic NFAT inhibition, which reduced both CX3CL1 and VCAM1 expression, significantly reduced TNFα-induced PBMC adhesion, and additional studies have shown that treatments directly targeting E-Selectin and ICAM1 also reduce the ability of leukocytes to adhere to the monolayer. Based on this, it was hypothesized that transfecting monolayers with siRNA specific for NFAT isoforms shown to reduce TNFα-induced expression of these genes would lead to decreases in TNFα-induced PBMC cell adhesion. Accordingly, transfection with NFATc2- and NFATc4-directed siRNA was found to reduce TNFα-induced PBMC cell adhesion. One might expect that conversely,

41

transfection with NFATc1 and NFATc3 siRNA, which increased TNFα-induced expression of these targets, might increase TNFα-induced PBMC adhesion, but neither NFATc1 nor NFATc3 knockdown had an effect on PBMC adhesion. In the case of NFATc1 siRNA this is likely due to the observed effect on ICAM1 expression being of minimal influence in the whole of the assay, and in the case of NFATc3, which had significant effects on both CX3CL1 and SELE expression, there being little room at the upper threshold of the assay for significant increases to TNFα-induced PBMC adhesion.

Collectively, these studies show a clear role for NFAT signaling, particularly NFATc2 and NFATc4, in the response of HRMEC to TNFα. Additionally, the isoform-specific effects identified in this study highlight the unique contributions of individual NFAT isoforms, as well as the need to target them individually.

42 CHAPTER IV

The Role of NFAT signaling in VEGF-Treated Human Retinal Microvascular Endothelial Cells

4.1 Overview

The previous studies examined the role of NFAT signaling in TNFα-induced retinal leukostasis (Chapter II & III), a pathogenic feature of early DR that contributes to microvascular complications and disease progression. An important aspect of this disease progression is the development of retinal neovascularization, which defines PDR, and can develop as a result of vascular complications or occlusions caused by leukostasis. VEGF, a powerful regulator of vascular permeability in early NPDR and DME, is also a critical driver of this late neovascular pathology in PDR, and therapeutics targeting the VEGF-signaling pathway have become the primary form of clinical treatment.

VEGF is a known inducer of NFAT activity in endothelial cells, and the present study evaluates the potential contributions of this activity to retinal neovascularization and PDR. Immunocytochemistry was initially used to confirm that VEGF stimulates NFAT nuclear translocation, a surrogate measure of NFAT activity, in HRMEC. After which, the inhibitor INCA-6 was used to evaluate the effect of NFAT blockade on HRMEC proliferation and tube formation in vitro. Lastly, the functional effects of pharmacologic NFAT inhibition were evaluated in vivo using a rat model of oxygen-induced retinopathy. NFAT inhibition reduced neovascularization in this context, highlighting an additional role for NFAT signaling in retinal neovascularization and PDR pathology.

43 4.2 Results